The process of hybridization of nucleic acids and is the formation of double stranded nucleic acids from single stranded nucleic acids, by specific interaction of complementary base pairs on the respective nucleic acid strands. Nucleic acids are long chain molecules made up of the nucleosides adenosine (A), guanosine (G), cytidine (C), and thymidine (I) in DNA or uridine (U) in RNA, covalently linked by phosphate ester linkages between the 3′-hydroxyl group on the sugar residue of one nucleoside and the phosphate linked to the 5′ position of the adjacent nucleoside, in various sequences. There is chemical affinity between respective pairs of purine and pyrimidine bases comprising part of each nucleotide unit (T-A and C-G). Hybridization thus requires the presence of complementary sequences of bases in nucleic acids, and is an extremely specific and sensitive reaction. It is the basis of oligonucleotide and DNA/RNA microarrays, such as biosensors, genosensors, or gene chips, being developed for use in the areas of genetic testing and sequencing, and drug discovery and development.
A critical component in obtaining genetic information is the ability to screen a DNA sequence. The microarray is a device which allows small scale and relatively high throughput nucleic acid analysis. Microarray analysis represents an aggregation of technologies, such as sequencing by hybridization (SBH), light-directed, spatially addressable interrogation, combinatorial chemical synthesis, confocal fluorescence microscopy, robotic spotting and polymerase chain reaction (PCR). These technologies offer significant advantages in terms of the avoidance of time-consuming protocols through the use of highly multiplexed analyses of DNA sequences.
The principle of SBH is based on the fact that a solution of digested linear DNA sequence incorporating the four bases is composed of overlapping shorter sequences. Accordingly, SBH employs hybridization of a set of single strand oligonucleotides or DNA with sub-sequences in a particular DNA fragment. In practical terms, the DNA target labelled with fluorescent tagging agents is allowed to interact with probes, which are attached to a substrate surface such as glass. Detection of the level of hybridization is effected using confocal fluorescence microscopy.
Microarrays are devices involving the attachment or immobilization of a known nucleic acid sequence (or “probe”) on a surface. Sensors for the detection of hybridization can be constructed from acoustic wave, optical and electrochemical devices. Detection of binding of a target nucleic acid from a test sample is effected by various methods such as radiolabelling, fluorescence or confocal microscopy. Once they have been exposed to the target (oligo)nucleic acids, some or most of the probes are hybridized as double-stranded nucleic acid or oligonucleotides. Conversion of double stranded nucleic acid to single stranded nucleic acid (denaturation) takes place relatively easily in aqueous solution using conventional methods such as heating, exposure to very high pH, of by using chemical denaturation, for example by exposure to urea. Different considerations apply to immobilized nucleic acid probes. Heating and chemical reagents have proven unsuccessful in the concurrent denaturation of double stranded DNA and regeneration of the surface-attached probe for re-use of the microarray. Accordingly, conventional microarrays are used once for a hybridization analysis and discarded thereafter. This makes microarray analysis of nucleic acids very expensive.
There are two methods for the fabrication of nucleic acid microarrays. The first involves the use of directed light via a combination of photolithography with combinatorial chemical synthesis. Bases are gradually added to a surface in a linear sequence by a photolithographical stepwise process, which includes the use of photolabile protecting moieties. Such a protocol can produce many thousands of probe nucleic acid sequences (approximately 20-mers) on a substrate surface on which the exact location of a particular probe is know accurately. The second approach involves attachment of a probe nucleic acid sequence by robotic spotting of the probe onto the surface of a substrate.
The pervasive strategy for the practical use of nucleic acid microarrays to date has been to discard the microarray after a single analysis. There are two significant disadvantages to this approach. First, despite much promise to the contrary, microarrays are still expensive to produce and purchase. Accordingly, if multiple analyses are to be performed on a particular sample the whole protocol becomes inordinately expensive. Second, particularly with respect to microarrays generated by robotic spotting of a probe onto a surface, it has proven difficult to produce identical microarrays that yield highly reproducible analytical results. Considerable variety in the pattern of probe density is often experienced leading to irreproducibility.